The second novel finding is that

The second novel finding is that La induces oxidative stress within C2C12 myoblasts. Our observations clearly demonstrate that 2h incubation with 20mM La DM induces 8-epi-PGF2α levels to rise. Adding AA, NAc, or LA to the 20mM La DM leads to a reduction of 8-epi-PGF2α levels, additionally establishing that ROS formation by La can be reversed by the use of antioxidants. Interestingly, the areas around the nuclei were mostly affected by oxidative stress where mitochondrial density is highest within the cell. We therefore additionally argue that La leads to increased ROS formation mostly within the mitochondrial membranes. This notion is supported by our finding that AA and NAc showed the highest efficacy of ROS scavenging. AA and NAc, in contrast to LA, unfold their direct antioxidative capacity mainly in the mitochondrion (Banaclocha, 2001; Nordberg and Arnér, 2001; Moreira et al., 2007; Mandl et al., 2009; Gillissen, 2011). Both substances were able to reduce the oxidative stress even below control levels. The mechanism by which LA deploys its antioxidative capacity remains elusive, but it has been described to act by the upregulation of the antioxidative enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (Cat) (Yu et al., 2013) which suggests a time-delayed, and hence a weakened effect for the time period observed. Nonetheless, further experiments are necessary to provide clear evidence of the idea that mitochondrial ROS generation is essentially elevated by La. The La-induced formation of ROS is controversially discussed in the literature. On the one hand, La was shown to be a capable scavenger of ROS in the absence of LDN193189 (Anbar and Neta, 1967; Groussard et al., 2000; Lampe et al., 2009). Whereas this finding was shown to be also present in cultured hepatocytes (Groussard et al., 2000), a protective effect was not established in neuronal precursor cells (Lampe et al., 2009). In contrast, other studies demonstrated that La increases ROS formation. One report describes the La-dependent enhancement of hydroxyl radical generation by the Fenton-reaction in a cell-free model (Ali et al., 2000). Hashimoto et al. (2007) showed an increased production of H2O2 in L6 myoblasts by 20mM La incubation indicating increased oxidative stress which is in agreement with our findings. Furthermore, the results for act. Casp-3 imply that La is able to induce apoptotic events, i.e. cellular stress. Whereas no data on the ability of La to induce the apoptotic pathway in C2C12 exists, ROS have been described to be able to initiate programmed cell death in this type of cells (Nishida et al., 2007; Gilliam et al., 2012; Lee et al., 2013). Our results unambiguously show that with La treatment, the oxidative stress increases within the cells. Therefore the rise in cleaved act. Casp-3 within cells is not surprising and provides further evidence for the conclusion that La induces cellular stress, marked by the increased generation of ROS. Thirdly we can report that La-induced effects are ROS-dependent and can therefore be reversed by the addition of antioxidants such as AA, NAc, and LA. Although no data is available on the effects of La on C2C12 differentiation, numerous studies exist elucidating the question how ROS influence this process. One report shows that the ROS-induced activation of NF-κB/iNOS pathway is necessary for differentiation (Piao et al., 2005). Another report from the same group implies that certain endogenous ROS concentrations are essential in differentiating myoblasts and that the addition of antioxidants to the cells resulted in differentiation inhibition. In contrast, Cyclosporin A-induced ROS generation has been shown to block myoblast differentiation in early myoblasts. The conclusion was that the addition of Cyclosporin A to differentiating cells led to toxic levels of ROS, blocking muscle differentiation even when antioxidants were added (Hong et al., 2002). Other studies provide evidence for a negative effect of ROS on myoblast differentiation. Langen et al. described that oxidative stress per se is sufficient to block myogenic late differentiation (Langen et al., 2002). H2O2 administered in different concentration (20–200μM) reduced myogenin and MHC protein content, creatine kinase activity as well as troponin I gene transcription all in a dose-dependent manner and that the inhibition of myotube formation was reversible when NAc was added to the culture medium (Langen et al., 2002). Furthermore it was demonstrated that 25μM H2O2 markedly reduced Myf5 and muscle regulatory factor 4 gene expression 2–3-fold, whereas myogenin was even 60-fold down-regulated (Hansen et al., 2007). This clearly supports the idea that myogenin expression, and therefore late myogenesis is very susceptible to oxidative stress, and thus La-sensitive. Applying 1mM H2O2 to C2C12 cells decreases Myf5 and MHC gene expression (Furutani et al., 2009). Taken together, the literature supports the notion that H2O2 can have different outcomes depending on the dose (Powers et al., 2010). However, regarding the data available for late differentiation markers as well as the findings on the reversibility of the La-effect by the addition of anti-oxidants, we conclude that the La-induced timely delay of late differentiation is mediated via ROS.